Project Summary The broad goal of this research is to improve (i) the potency, (ii) safety, and (iii) the credibility of nerve stimulation as a treatment for sepsis by defining the functional neural circuitry. In the USA, among 1-3 million patients suffering sepsis each year, 15-30% will die and many of the survivors will suffer organ damage. Conventional therapeutic regimens are thus far inadequate. Against this backdrop, nerve stimulations such as non-invasive electroacupuncture stimulation (ES) at specific acupoints can attenuate systemic inflammation associated with sepsis and promote survival of laboratory animals. To date, one dominant view is that ES drives vagal nerve-dependent anti-inflammatory pathways, involving activation of sympathetic cells and subsequent modulation of pro-inflammatory cytokine release from immune cells. However, there are two important unresolved issues that must be addressed in order to realize the full potential of ES as a therapeutic modality for sepsis. First, the roles of sympathetic neurons are still ill-defined. Noradrenaline (NA), one of transmitters released from sympathetic cells, has long been proposed to suppress systemic inflammation, via activation of ?2 adrenergic receptors. However, NA can also promote inflammation via activation of ?2 adrenergic receptors, and this pro-inflammatory signaling can be sensitized following macrophage pre- exposure to bacteria-derived endotoxins such as the lipopolysaccharide (LPS). Accordingly, it remains unknown i) if ES could be counterindicated when sepsis has progressed to certain stages, and ii) if a strategy to maximize the anti-inflammatory over the pro-inflammatory activity is pivotal for ES to treat severe sepsis. Second, it has been long known that ES can drive vagal parasympathetic reflexes only in specific acupoints, but the underlying neural basis is entirely unknown. Because of unknown identities and anatomical distributions of somatosensory neurons driving vagal reflexes, it becomes difficult to optimize stimulation parameters to activate this anti-inflammation pathway. To address these unresolved issues, we have developed innovative genetic tools to ablate, silence or activate molecularly defined sympathetic and somatosensory neurons. Built upon strong preliminary results, we postulate i) that sensory neurons marked by the expression of the G protein-coupled receptor Prokr2 are required to for low electric intensity ES (0.5 mA) to drive vagal reflexes, and ii) that noradrenergic sympathetic neurons marked by the expression of the neuropeptide NPY, which can be activated by high intensity ES (3 mA), may suppress and promote inflammation, dependent on ES delivered before or after sepsis manifestation. A series of predictions from these hypotheses will be tested. In the fullness of time, the studies outlined in this application will enable us to illustrate distinct neuronal pathways that can dynamically modulate sepsis-associated systemic inflammation, and will help to improve sepsis management by promoting the anti-inflammatory over the pro-inflammatory pathways. !